EP2379711B1 - Zielpopulationen von oligodendrozyten-vorläuferzellen und verfahren zur herstellung und verwendung davon - Google Patents

Zielpopulationen von oligodendrozyten-vorläuferzellen und verfahren zur herstellung und verwendung davon Download PDF

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EP2379711B1
EP2379711B1 EP09795884.7A EP09795884A EP2379711B1 EP 2379711 B1 EP2379711 B1 EP 2379711B1 EP 09795884 A EP09795884 A EP 09795884A EP 2379711 B1 EP2379711 B1 EP 2379711B1
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cells
opcs
population
pdgfrα
neural
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EP2379711A1 (de
EP2379711B8 (de
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Alexandra Capela
Nobuko Uchida
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Bright Ocens Silicon Valley Inc
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StemCells California Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/135—Platelet-derived growth factor [PDGF]

Definitions

  • This invention relates to isolation, characterization, proliferation, differentiation and transplantation of a population of oligodendrocyte precursor cells.
  • neural stem cells During development of the central nervous system (“CNS”), multipotent neural precursor cells, also known as neural stem cells, proliferate and give rise to transiently dividing progenitor cells that eventually differentiate into the cell types that compose the adult brain. Stem cells (from other tissues) have classically been defined as having the ability to self-renew ( i.e. , form more stem cells), to proliferate, and to differentiate into different phenotypic lineages. In the case of neural stem cells, this includes neurons, astrocytes and oligodendrocytes. Neural stem cells have been isolated from several mammalian species, including mice, rats, pigs and humans.
  • oligodendrocytes The main function of oligodendrocytes is the myelination of axons in the central nervous system of higher vertebrates. Oligodendrocyte precursor cells (OPCs) precede oligodendrocytes.
  • OPCs Oligodendrocyte precursor cells
  • the invention relates to enriched target populations of oligodendrocyte precursor cells (OPCs) that can further differentiate into oligodendrocytes.
  • OPCs oligodendrocyte precursor cells
  • the invention provides an enriched population of OPCs derived from neural or neural derived cells wherein the population is enriched for OPCs that are PDGFR ⁇ + and CD 105 - and A2B5 lo/- , wherein at least 30% of the cells in the population are PDGFR ⁇ + and CD105 - and A2B5 lo/- , wherein the OPCs in the population are committed to differentiation into mature oligodendrocytes under conditions that allow differentiation,
  • the target populations of cells are enriched for OPCs that are immunopositive for PDGFR ⁇ (PDGFR ⁇ - ), immunopositive for CD133 (CD133 + ), and immunonegative for CD105 (CD105 - ).
  • the target OPCs are PDGFR ⁇ + , CD105 - and additionally A2B5 lo , A2B5 - , or mixture thereof (A2B5 lo/- ).
  • the target populations of cells are enriched for OPCs that are immunopositive for PDGFR ⁇ (PDGFR ⁇ + ), immunopositive for CD133 (CD133 + ), immunonegative for A2B5 (A2B5 - ), and immunonegative for CD105 (CD105 - ).
  • the target population of OPCs are PDGFR ⁇ + , CD105 - , A2B5 lo/- .
  • the target population of OPCs are PDGFR ⁇ - , CD133 + , CD105 - , A2B5 - .
  • the target population of OPCs are PDGFR ⁇ + , CD133 - , CD105 - , A2B5 lo/- .
  • the target population of OPCs arc PDGFR ⁇ + , CD133 + , CD105 - , A2B5 - , PSA-NCAM - .
  • the target population of OPCs are PDGFR ⁇ + , CD133 + , CD105 - , A2B5 lo/- , PSA-NCAM - .
  • the target population of OPCs are PDGFR ⁇ + , CD133 - , CD105 - , A2B5 - , PSA-NCAM lo/- .
  • the target population of OPCs are PDGFR ⁇ + , CD133 + , CD105 - , A2B5 lo/- , PSA-NCAM lo/- .
  • the invention provide in vitro methods for producing populations enriched for target oligodendrocyte precursor cells by contacting neural or neural derived cells with at least one antibody that binds to CD105; selecting the cells that bind to this antibody; and removing the bound cells and contacting the remaining cells with at least one antibody that binds to PDGFR ⁇ .
  • the remaining cells in the population are enriched for oligodendrocyte precursor cells as defined above.
  • a negative selection marker is a marker (i.e . an antigen) that is present only on non-OPC cells.
  • the antibody may be a monoclonal antibody which may be fluorochrome conjugated or may be conjugated to magnetic particles, and the selection may be by fluorescence activated cell sorting, high gradient magnetic selection, by attachment to and disattachmcnt from the solid phase, or any other commonly used selection technique.
  • the population containing neural or neural-derived cells is obtained from a suspension culture, an adherent culture, or from fresh neural tissue. These methods may also involve the step of further enriching the population for oligodendrocyte precursor cells by contacting the remaining cells with a further antibody or series of antibodies.
  • target populations of OPCs may be enriched by contacting the culture of neural or neural derived cells with an antibody that specifically binds to CD133 followed by contacting the remaining cells with an antibody that specifically binds PDGFR ⁇ to produce populations enriched for oligodendrocyte precursor cells that are immunopositive for both CD133 and PDGFR ⁇ .
  • the neural or neural derived cells can be obtained from a neurosphere culture (cell clusters or cell aggregates) or from an adherent culture.
  • the method also involves the step of eliminating those cells that are PDGFR ⁇ lo/med from the population.
  • the invention therefore relates to methods for isolating a oligodendrocyte precursor cell (OPC), by selecting from a population of neural or neural-derived cells for cells that are immunonegative for CD105 (CD105 - cells); eliminating the immunoreactive (CD105 + ) cells from the population; and selecting from the remaining population for at least one cell that is immunopositive for PDGFR ⁇ (PDGFR ⁇ - ), e.g. , binds to monoclonal antibody PDGFR ⁇ .
  • OPC oligodendrocyte precursor cell
  • oligodendrocyte precursor cell is isolated by selecting from a population of neural or neural-derived cells for cells that are immunopositive for PDGFR ⁇ (PDGFR ⁇ + ), e.g ., binds to monoclonal antibody PDGFR ⁇ ; eliminating the non-immunoreactive (PDGFR ⁇ - ) cells from the population; and selecting from the remaining population for at least one cell that is immunonegative for CD105 (CD105 - ).
  • oligodendrocyte precursor cells by contacting neural or neural derived cells containing at least one multipotent neural stem cell with an antibody that specifically binds to PDGFR ⁇ and selecting those cells that are PDGFR ⁇ hi , wherein the selected cells are enriched for oligodendrocyte precursor cells as compared with the neural or neural derived cells.
  • the neural or neural derived cells can be obtained from a neurosphere culture or from an adherent culture. In some embodiments, this method also involves the step of eliminating those cells that are PDGFR ⁇ lo/med from the population.
  • oligodendrocyte precursor cells by eliminating cells that are positive for markers of differentiated cells or fibroblasts cells from a population of neural or neural-derived cells (e.g ., CD105). This may be accomplished by contacting the population with a monoclonal antibody directed to such markers of differentiated cells and removing those cells that bind to this monoclonal antibody. This resulting population of cells may be further enriched using any of the methods described herein.
  • the method may involve the step of further enriching the population for oligodendrocyte precursor cells by contacting the remaining cells with an antibody that specifically binds to PDGFR ⁇ .
  • the fraction may optionally be enriched by selecting from the remaining cells for cells are PDGFR ⁇ + , CD105 - , CD133 + , A2B5 lo/- , PSA-NCAM lo/- and mixtures and combinations thereof.
  • the invention provides methods for proliferating oligodendrocyte precursor cells by proliferating a population of OPCs in serum free culture medium containing one or more growth factors effective for inducing OPC proliferation, wherein the growth factor comprises LIF, EFG, FGF-2, bFGF, GF-1, NT3, Shh, CTNF, PDGF, PDGF-AA, or combinations thereof, and wherein the population comprises OPCs which are PDGFR ⁇ + , CD105- and A2B5 lo/- .
  • the induction of proliferation (and differentiation) of the OPCs can be done either by culturing the cells in suspension or on a substrate onto which they can adhere.
  • proliferation and differentiation of OPCs can be induced, under appropriate conditions, in the host in the following combinations: (1) proliferation and differentiation in vitro , then transplantation, (2) proliferation in vitro , transplantation, then further proliferation and differentiation in vivo, (3) proliferation in vitro, transplantation and differentiation in vivo , and (4) proliferation and differentiation in vivo .
  • Proliferation and differentiation in vivo or in situ can involve a non-surgical approach that coaxes OPCs to proliferate in vivo with pharmaceutical manipulation.
  • the invention provides an enriched population of OPCs for use in treating or ameliorating a demyelinating or dysmyelinating disease or disorder in a mammal.
  • the mammal preferably harbors a demyelinating or dysmyelinating disease, including, but not limited to, multiple sclerosis, acute disseminated encephalomyelitis, diffuse cerebral sclerosis, necrotizing hemorrhagic encephalitis, radiation induced myelination disorders, transverse myelitits, Pelizaeus-Merzbacher disease (PMD), Cerebral palsy (CP), and leukodystrophies.
  • the disease is preferably multiple sclerosis, Pelizaeus-Merzbacher disease, or Cerebral palsy.
  • the mammal may additionally receive at least one biological agent that is capable of increasing the number of OPCs and/or at least one factor that is known to stimulate oligodendrocyte differentiation, growth, proliferation, or survival.
  • the OPCs for use may be administered in any manner that results in contact of the factor and or agent with target OPCs in the mammal, such as systemically ( e.g. , subcutaneously) or in situ .
  • the OPCs for use may be administered into the brain, more preferably into the lateral ventricle of the brain or into the brain parenchyma.
  • target cell population denotes those cells which are desirably being purified or enriched.
  • the target cell population are oligodendrocyte precursor cells that display the distinctive pattern of cell markers as described herein.
  • OPC central nervous system
  • neural stem cells is the more general term used for undifferentiated, multipotent, self-renewing, neural cells.
  • a neural stem cell is a clonogenic multipotent stem cell which is able to divide and, under appropriate conditions, has self-renewal capability and can include in its progeny daughter cells which can terminally differentiate into neurons, astrocytes, and oligodendrocytes.
  • the neural stem cell is "multipotent” because stem cell progeny have multiple differentiation pathways.
  • a neural stem cell is capable of self maintenance, meaning that with each cell division, one daughter cell will also be a stem cell.
  • the non-stem cell progeny of a neural stem cell are typically referred to as "progenitor” or “precursor” cells, which are capable of giving rise to various cell types within one or more lineages.
  • the term "neural progenitor cell” or “neural precursor cell” refers to an undifferentiated cell derived from a neural stem cell and is not itself a stem cell.
  • Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type.
  • an O-2A cell is a glial progenitor cell that gives rise to oligodendrocytes and type II astrocytes, and, thus, could be termed a "bipotential" progenitor cell.
  • a distinguishing feature of a progenitor cell is that, unlike a stem cell, it does not exhibit self maintenance. Moreover, progenitor cells are typically thought to be committed to a particular path of differentiation and will, under appropriate conditions, eventually differentiate into glia or neurons.
  • neural cells refers broadly to cells associated with the central nervous system (CNS) of an organism, for example, neurons, glial cells, and precursor cells.
  • CNS central nervous system
  • neural cells may be cells that are isolated or derived from neural tissue, as well as any cell, regardless of origin, having at least an indication of neuronal or glial phenotype, such as staining for one or more neuronal or glial markers or which will differentiate into cells exhibiting neuronal or glial markers.
  • neural cells may be used as a general term to refer to, for example, primary cells isolated and cultured in vitro ; cultured immortalized cells derived from a neural tissue, neural tissue cells; and/or cells cultured to express a neural phenotype.
  • the term is meant to be all-encompassing with respect to cells exhibiting a neural cell phenotype and/or isolated from neural tissue.
  • neural cells also includes cells which are neural precursor cells as well as differentiated neural cells.
  • neuroonal cells refers to neurons.
  • positive selection refers to a process in which the target cell population is purified or enriched by removing the target cell population from a mixture of cell populations by directly binding the target cell population to reagents having affinity therefore.
  • the term "negative selection” refers to a process in which the target cell population is purified or enriched by removing nontarget cell populations from the mixture of cells by binding the nontarget cell populations to reagents having affinity therefore.
  • CD45 is the T200/leucocyte common antigen.
  • CNS-SC Human central nervous system stem cells
  • reagents that recognize CD45 may be useful in a negative selection process to remove nontarget cells.
  • a "primary neurosphere” is a neurosphere generated by culturing brain tissue. Typically, the brain tissue is dissected and mechanically dissociated before being cultured in appropriate media and allowed to form neurospheres. Exemplary methods are described in, for instance, U.S. Pat. No. 5,750,376 , the disclosure of which is incorporated herein by reference in its entirety.
  • a "secondary neurosphere” is a neurosphere generated by dissociating (passaging) a primary neurosphere and culturing the dissociated cells under conditions which result in the formation of neurospheres from single cells.
  • a "mammal” is any member in the mammalian family.
  • a mammal is preferably a primate, rodent, feline, canine, domestic livestock (such as cattle, sheep, goats, horses, and pigs), and most preferably a human.
  • a "demyelinating disease” or a “dysmyelinating disorder” is a disease, disorder, or medical condition that is caused by or associated with inadequate amounts myelin.
  • Demyelination is the process of myelin removal i.e. , loss of myelin that existed before.
  • Dysmyelination occurs where no or inadequate amounts of myelin forms, e.g ., due to dysfunctional OPCs or oligodendrocytes (OLs). The end result of demyelination and dysmyelination is hypomyelination.
  • diseases, disorders, or conditions include, for example, multiple sclerosis (including the relapsing and chronic progressive forms of multiple sclerosis, acute multiple sclerosis, neuromyelitis optica (Devic's disease)), diffuse cerebral sclerosis (including Shilder's encephalitis periaxialis diffusa and Balo's concentric sclerosis).
  • Demyelinating diseases or dysmyelinating disorders also include a variety of diseases wherein demyelination is caused by viral infections, vaccines, spinal cord injury, and genetic disorders.
  • demyelinating diseases or dysmyelinating disorders include acute disseminated encephalomyelitis (occurring after measles, chicken pox, rubella, influenza or mumps; or after rabies or small pox vaccination), necrotizing hemorrhagic encephalitis (including hemorrhagic leukoencephalitis), and leukodystrophies (including Krabbe's globboid leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, adrenonryeloneuropathy, adrenomyeloneuropathy, radiation induced myelination disorders, transverse myelitits, Pelizaeus-Merzbacher disease (PMD), Canavan's disease and Alexander's disease).
  • acute disseminated encephalomyelitis occurring after measles, chicken pox, rubella, influenza or mumps; or after rabies or small pox vaccination
  • the demyelinating disease or dysmyelinating disorder is preferably multiple sclerosis, cerebral palsy, diffuse cerebral sclerosis, or Pelizaeus-Merzbacher disease (PMD), and, most preferably, Pelizaeus-Merzbacher disease.
  • PMD Pelizaeus-Merzbacher disease
  • Treating or “ameliorating” means the reduction or complete removal of the symptoms of a disease or medical condition.
  • an “effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose.
  • the effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration.
  • the effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.
  • ventricle refers to any cavity or passageway within the CNS through which cerebral spinal fluid flows. Thus, the term not only encompasses the lateral, third, and fourth ventricles, but also encompasses the central canal, cerebral aqueduct, and other CNS cavities.
  • This invention relates to the identification, isolation, enrichment, and culture of oligodendrocyte precursor cells.
  • the target cell population of OPCs can be characterized by their expression of cell surface markers. While it is commonplace in the art to refer to cells as “positive” or “negative” for a particular marker, actual expression levels are a quantitative trait. The number of molecules on the cell surface can vary by several logs, yet still be characterized as "positive”. It is also understood by those of skill in the art that a cell which is negative for staining, i.e ., the level of binding of a marker specific reagent is not detectably different from a control, e.g . an isotype matched control; may express minor amounts of the marker. Characterization of the level of staining permits subtle distinctions between cell populations.
  • the staining intensity of cells can be monitored by flow cytometry, where lasers detect the quantitative levels of fluorochrome (which is proportional to the amount of cell surface marker bound by specific reagents, e.g . antibodies).
  • Flow cytometry, or FACS can also be used to separate cell populations based on the intensity of binding to a specific reagent, as well as other parameters such as cell size and light scatter.
  • the absolute level of staining may differ with a particular fluorochrome and reagent preparation, the data can be normalized to a control.
  • each cell is recorded as a data point having a particular intensity of staining.
  • These data points may be displayed according to a log scale, where the unit of measure is arbitrary staining intensity.
  • the brightest cells in a population are designated as 4 logs ( i.e. , 10,000 times) more intense than the cells having the lowest level of staining.
  • the "low" staining cells which fall in the 2-3 log ( i.e ., 100-1000 fold) of staining intensity, may have properties that are unique from the negative and positive cells.
  • An alternative control may utilize a substrate having a defined density of marker on its surface, for example a fabricated bead or cell line, which provides the positive control for intensity.
  • the "low" designation indicates that the level of staining is above the brightness of an isotype matched control, but is not as intense as the most brightly staining cells normally found in the population.
  • an isotype matched control will define the signal intensity of "non-specific" or “negative” staining. Whereas any staining which results in signal intensity above that of the control is considered to be “positive” staining.
  • the boundary demarcating negative and positive staining is conventionally set such that the frequency of events to the left of, or below, the boundary is > 0.99 and ⁇ 1.0.
  • Positive staining intensity can then be further subdivided and categorized as low, medium, or high by defining an arbitrary scale from the control boundary to the highest recorded signal intensity and defining two additional lines of demarcation at the 33 rd and 66 th percentiles, respectively. Signals measured in the lower-third, middle-third, and upper-third of these defined groups can then be designated as low, medium, and high staining intensity, respectively.
  • cell surface antigens to isolate, select, or enrich for OPC cells provides a means for the positive and negative immunoselection of target OPC populations, as well as for the phenotypic analysis of target OPC cell populations using flow cytometry.
  • a subset of OPCs is separated from other cells on the basis of PDGFR ⁇ binding.
  • OPCs may be further separated by binding to other surface markers known in the art.
  • Cells selected for expression of PDGFR ⁇ antigen for example, may be further purified by the positive and negative immunoselection of other target OPC markers as disclosed herein.
  • Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography and "panning" with antibody attached to a solid matrix, e.g . plate, or other convenient technique.
  • Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • Dead cells may be eliminated by selection with dyes associated with dead cells (propidium iodide [PI], LDS). Any technique may be employed which is not unduly detrimental to the viability of the selected cells.
  • the antibodies are conjugated with labels to allow for ease of separation of the particular cell type, e.g . magnetic beads; biotin, which binds with high affinity to avidin or streptavidin; fluorochromes, which can be used with a fluorescence activated cell sorter; haptens; and the like.
  • Multi-color analyses may be employed with the FACS or in a combination of immunomagnetic separation and flow cytometry. Multi-color analysis is of interest for the separation of cells based on multiple surface antigens, e.g . PDGFR ⁇ + , CD105 - , CD133 + , CD24 - , etc.
  • Fluorochromes which find use in a multi-color analysis include, for example, phycobiliproteins, e.g . phycoerythrin and allophycocyanins; fluorescein and Texas red.
  • a negative designation indicates that the level of staining is at or below the brightness of an isotype matched negative control.
  • a "dim”, “lo”, or “low” designation indicates that the level of staining may be near the level of a negative stain, but may also be brighter than an isotype matched control.
  • the PDGFR ⁇ antibody is directly or indirectly conjugated to a magnetic reagent, such as a superparamagnetic microparticle (microparticle).
  • a magnetic reagent such as a superparamagnetic microparticle (microparticle).
  • Direct conjugation to a magnetic particle is achieved by use of various chemical linking groups, as known in the art.
  • Antibody can be coupled to the microparticles through side chain amino or sulfhydryl groups and heterofunctional cross-linking reagents. A large number of heterofunctional compounds are available for linking to entities.
  • a preferred linking group is 3-(2-pyridyidithio) propionic acid N-hydroxysuccinimide ester (SPDP) or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC) with a reactive sulfhydryl group on the antibody and a reactive amino group on the magnetic particle.
  • SPDP 3-(2-pyridyidithio) propionic acid N-hydroxysuccinimide ester
  • SMCC 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester
  • the antibody is indirectly coupled to the magnetic particles.
  • the antibody may be directly conjugated to a hapten, and hapten-specific, second stage antibodies are conjugated to the particles.
  • Suitable haptens include, for examples digoxin, digoxigenin, FITC, dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugation of the hapten to a protein, i.e. are known in the art, and kits for such conjugations are commercially available.
  • Target OPC populations are selected by bringing neural or neural-derived cells into contact with the antibody or reagent that binds the surface marker.
  • antibody is added to a cell sample.
  • the amount of antibody or other reagent necessary to bind a particular cell subset is empirically determined by performing a test separation and analysis.
  • the cells and antibody/reagent are incubated for a period of time sufficient for complexes to form, preferably at least about 5 min, more preferably at least about 10 min, and usually not more than one hr, more usually not more than about 30 min.
  • the cells may additionally be incubated with antibodies or binding molecules specific for cell surface markers known to be present or absent on OPCs.
  • the labeled cells are separated in accordance with the specific antibody preparation.
  • Fluorochrome labeled antibodies are useful for FACS separation, magnetic particles for immunomagnetic selection, particularly high gradient magnetic selection (HGMS), etc.
  • Exemplary magnetic separation devices are described in WO 90/07380 , PCT/US 96/00953 , and EP 438,520 , the disclosures of which are herein incorporated by reference in their entireties.
  • the purified cell population may be collected in any appropriate medium.
  • Various media are commercially available and may be used, including Dulbecco's Modified Eagle Medium (DMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (DPBS), RPMI, Iscove's modified Dulbecco's medium (IMDM), phosphate buffered saline (PBS) with 5 mM EDTA, etc. , frequently supplemented with fetal calf serum (FCS), bovine serum albumin (BSA), human serum albumin (HSA), etc .
  • FCS fetal calf serum
  • BSA bovine serum albumin
  • HSA human serum albumin
  • the desired cells will be 30% or more of the cell composition, preferably 50% or more (e.g ., 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more) of the cell population, more preferably 90% or more ( e.g. , 92% or more, 94% or more, 96% or more, or 98% or more) of the cell population, and most preferably 95% or more ( e.g ., 97%, 99%) (substantially pure) of the cell population.
  • the degree of enrichment obtained, and actually used depends on a number of factors, including, but not limited to, the method of selection, the method of growth, and/or the dose of the cells that are placed in culture.
  • the invention relates to the isolation and identification of OPCs.
  • the methods may be used to isolate PDGFR ⁇ + cells from PDGFRC ⁇ - cells using an PDGFR ⁇ antibody or other reagent that specifically binds to PDGFR ⁇ by combining a population of neural or neural-derived cells which contains a fraction of OPCs with the antibody or reagent, and then selecting for PDGFR ⁇ + cells, to produce a selected population enriched in PDGFR ⁇ - OPCs as compared with the population of neural or neural-derived cells before selection.
  • the population of cells from which OPCs are isolated is preferably a neural tissue, a population of cells dissociated from neural tissue, a population of cells that can give rise to neural cells or neural tissue, or a population of cells in cell culture, e.g ., cells in a neurosphere culture or an adherent neural stem cell culture.
  • Identification of oligodendrocyte precursor cell (OPC) involves contacting a population of cells or neural cells (or tissue which contains neural or neural-derived cells) with a reagent that binds to cell surface markers expressed by the target population of OPCs.
  • the method comprises contacting a population of neural or neural-derived cells with at least one antibody that binds to PDGFR ⁇ (e.g.
  • Target OPCs are included in the population of cells that reagent binds to the reagent. The identity of those cells can be confirmed by any assays known on the art to demonstrate that the cells are, in fact, OPCs, i.e. , capable of proliferation and capable of differentiating into mature oligodendrocytes.
  • the OPCs according to the invention are further characterized as being immunonegativc for CD105 (CD105 - ).
  • the method of identifying, isolating, or enriching populations of oligodendrocyte precursor cells comprises selecting from a population of neural or neural-derived cells for cells that are immunonegative for CD105 (CD105 - cells) and eliminating the CD105 + cells from the population. Selection of CD105 - cells may then be followed by selecting from the remaining population for at least one cell that is immunopositive for PDGFR ⁇ (PDGFR ⁇ + ).
  • the invention provides methods for isolating a oligodendrocyte precursor cell (OPC), by selecting from a population of neural or neural-derived cells for cells that are immunopositive for PDGFR ⁇ (PDGFR ⁇ + ), e.g. , binds to monoclonal antibody PDGFR ⁇ ; eliminating the non-immunoreactive (PDGFR ⁇ - ) cells from the population; and selecting from the remaining population for cells that are immunonegative for CD105 (CD105 - ), i.e. , eliminating CD105 + cells from the population.
  • PDGFR ⁇ + oligodendrocyte precursor cell
  • the OPCs according to some embodiments may be further characterized as being immunopositive for CD133 (CD133 + ).
  • the method of identifying, isolating, or enriching populations of oligodendrocyte precursor cells may additionally comprise selecting from a population of neural or neural-derived cells for cells that are immunopositive for CD133 (CD133 + cells) and eliminating the CD133 - cells from the population. Selection of CD133 + cells may then be followed by selecting from the remaining population for at least one cell that is immunopositive for PDGFR ⁇ (PDGFR ⁇ + ).
  • the invention further provides for the enrichment of target OPCs from neural tissue or neural stem cell cultures (e.g ., suspension cultures or adherent cultures).
  • neural tissue or neural stem cell cultures e.g ., suspension cultures or adherent cultures.
  • the methods and composition of the invention are thus useful for the enrichment of target OPC from neural tissue in which stem cells and progenitor cells occur at low frequency, or may have been depleted, such as late embryo, juvenile, and adult tissue.
  • a reagent that specifically binds to, for example, PDGFR ⁇ , and then select for the PDGFR ⁇ + cells.
  • the selected PDGFR ⁇ + cells are enriched in the fraction of OPC as compared with the population of neural or neural-derived cells.
  • the target OPCs may be characterized based on a medium to high expression of PDGFR ⁇ (e.g. , PDGFR ⁇ med or PDGFR ⁇ high ).
  • target OPCs are included in the PDGFR ⁇ + cells sorted from suspended neurospheres based on medium to high expression ( e.g ., PDGFR ⁇ med or PDGFR ⁇ high ).
  • Target OPCs may be further identified by their expression of the markers CD105, CD133, A2B5, PSA-NCAM, 04, and/or NG2 in accordance with the present invention.
  • any method for selecting a population of cells on the basis of cell marker expression known in the art may be used to select for the OPCs of the present invention.
  • the identification of PDGFR ⁇ + and/or CD133 + target cell populations may involve contacting a population of neural cells (or tissue which contains neural or neural derived cells) with a reagent that binds to PDGFR ⁇ and/or CD133, and detecting the contact between the reagent that binds to PDGFR ⁇ and/or CD133 and PDGFR ⁇ and/or CD133 on the surface of cells.
  • Target OPCs are included in the population of those cells to which the reagent binds.
  • those cells can be confirmed by assays to demonstrate that the cells are, in fact, OPCs, i.e ., they are capable of differentiating into mature oligodendrocytes.
  • FACS fluorescence activated cell separation
  • One of skill in the art can introduce an isolated target OPC(s) to a culture medium; proliferate the isolated target OPC(s) in culture; culture the progeny of the isolated target OPC(s) under conditions in which the isolated target OPC(s) differentiates into oligodendrocytes; and detect the presence of oligodendrocytes.
  • the presence of oligodendrocytes characterizes the isolated target OPC(s) as an OPC.
  • any cell markers known in the art may also be used for the positive and negative selection of OPCs.
  • monoclonal antibodies (mAb) against human CD45 may be used to exclude blood cell contamination in fetal tissue.
  • mAb against human CD34 may be used to exclude endothelial cells and endothelial-neural progenitor complexes.
  • antibodies against human CD24 may be used to exclude those cells that are not likely to initiate neurospheres. Any of these antibodies may be used alone, in combination, or sequentially in the methods for enriching the target cell populations disclosed herein.
  • the population of target cells contains at least 30% PDGFR ⁇ + OPCs, preferably at least 50-70% PDGFR ⁇ + OPCs, and more preferably greater than 90% PDGFR ⁇ + OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ + OPCs, comprising at least 95% PDGFR ⁇ + OPCs ( e.g ., 97% or 99%).
  • the population of target cells contains at least 30% PDGFR ⁇ + , CD105 - OPCs, preferably at least 50-70% PDGFR ⁇ + , CD105 - OPCs, and more preferably greater than 90% PDGFR ⁇ + , CD105 - OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ + , CD105 - OPCs, comprising at least 95% PDGFR ⁇ + , CD105 - OPCs ( e.g. , 97% or 99%).
  • the population of target cells contains at least 30% PDGFR ⁇ + , A2B5 - OPCs, preferably at least 50-70% PDGFR ⁇ + , A2B5 - OPCs, and more preferably greater than 90% PDGFR ⁇ + , A2B5 - OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ + , A2B5 - OPCs, comprising at least 95% PDGFR ⁇ + , A2B5 - OPCs ( e.g ., 97% or 99%).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ + , A2B5 lo/- OPCs, preferably at least 50-70% PDGFR ⁇ + , A2B5 lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ + , A2B5 lo/- OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ + , A2B5 lo/- OPCs, comprising at least 95% PDGFR ⁇ + , A2B5 lo/- OPCs ( e.g ., 97% or 99%).
  • the population of target cells is additionally immunonegative for CD 105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ med/high , A2B5 - OPCs, preferably at least 50-70% PDGFR ⁇ med/high , A2B5 - . OPCs, and more preferably greater than 90% PDGFR ⁇ med/high , A2B5 - . OPCs ( e.g. , 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ med/high , A2B5 - . OPCs, comprising at least 95% PDGFR ⁇ med/high , A2B5 - . OPCs ( e.g ., 97% or 99%).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ med/high , A2B5 lo/- OPCs, preferably at least 50-70% PDGFR ⁇ med/high , A2B5 lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ med/high , A2BS lo/- OPCs ( e.g. , 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ med/high , A2B5 lo/- OPCs, comprising at least 95% PDGFR ⁇ med/high , A2B5 lo/- OPCs ( e.g ., 97% or 99%).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ + , PSA-NCAM - OPCs, preferably at least 50-70% PDGFR ⁇ + , PSA-NCAM - OPCs, and more preferably greater than 90% PDGFR ⁇ + , PSA-NCAM- OPCs ( e.g. , 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ + , PSA-NCAM - OPCs, comprising at least 95% PDGFR ⁇ + , PSA-NCAM - OPCs ( e.g ., 97% or 99%).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ + , PSA-NCAM lo/- OPCs, preferably at least 50-70% PDGFR ⁇ + , PSA-NCAM lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ + , PSA-NCAM lo/- OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ + , PSA-NCAM lo/- OPCs, comprising at least 95% PDGFR ⁇ + , PSA-NCAM lo/- OPCs ( e.g ., 97% or 99%).
  • the population of target cells is additionally immunonegative for CD 105 (CD 105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ med/high , PSA-NCAM - OPCs, preferably at least 50-70% PDGFR ⁇ med/high , PSA-NCAM - OPCs, and more preferably greater than 90% PDGFR ⁇ med/high , PSA-NCAM - OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ med/high , PSA-NCAM - OPCs, comprising at least 95% PDGFR ⁇ med/high , PSA-NCAM - OPCs ( e.g ., 97% or 99%).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ med/high , PSA-NCAM lo/- OPCs, preferably at least 50-70% PDGFR ⁇ med/high , PSA-NCAM lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ med/high , PSA-NCAM lo/- OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ + , CD133 + , A2B5 lo/- OPCs, preferably at least 50-70% PDGFR ⁇ + , CD133 + , A2B5 lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ + , CD133 + , A2B5 lo/- OPCs ( e.g. , 92% or more, 94% or more, 96% or more, or 98% or more).
  • the population of target cells is additionally immunonegative for CD105 (CD 105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ + , CD133 + , PSA-NCAM lo/- OPCs, preferably at least 50-70% PDGFR ⁇ + , CD133 + , PSA-NCAM lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ + , CD133 + , PSA-NCAM lo/- OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ + , A2B5 lo/- , PSA-NCAM lo/- OPCs, preferably at least 50-70% PDGFR ⁇ + , A2B5 lo/- , PSA-NCAM lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ + , A2B5 lo/- , PSA-NCAM lo/- OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ med/high , A2B5 lo/- , PSA-NCAM lo/- OPCs, preferably at least 50-70% PDGFR ⁇ med/high , A2B5 lo/- , PSA-NCAM lo/- OPCs, and more preferably greater than 90% PDGFR ⁇ med/high , A2B5 lo/- PSA-NCAM lo/- OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more).
  • the population of target cells is additionally immunonegative for CD105 (CD15 - ).
  • the population of target cells contains at least 30% PDGFR ⁇ + , O4 - OPCs, preferably at least 50-70% PDGFR ⁇ + , O4 - OPCs, and more preferably greater than 90% PDGFR ⁇ + , O4 - OPCs ( e.g ., 92% or more, 94% or more, 96% or more, or 98% or more). Most preferable would be a substantially pure population of PDGFR ⁇ + , O4 - OPCs, comprising at least 95% PDGFR ⁇ + , O4 - OPCs ( e.g ., 97%, 99%).
  • the population of target cells is additionally immunonegative for CD105 (CD105 - ). The degree of enrichment obtained, and actually used, depends on a number of factors, including the method of selection, the method of growth, and/or the dose of the cells that are placed in culture.
  • the OPCs of the invention may be cryopreserved according to routine procedures.
  • cryopreserving involves freezing about one to ten million cells in "freeze" medium, which may comprise proliferation medium and antioxidants such as NAC (0.1 to 2mM; e.g. , 0.5 mM, 1mM, etc.).
  • Proliferation medium is preferably absent the growth factor mitogens.
  • suspended cells may be centrifuged and any growth medium is aspirated and replaced with freeze medium. Cells may then be slowly frozen, by, e.g. , placing in a container at -80° C or frozen in liquid nitrogen. Cells are thawed by swirling in a 37" C bath, resuspended in fresh proliferation medium, and grown as usual.
  • the OPCs of the present embodiments may be cryopreserved in a ready to use format (such as a pharmaceutical grade vial or container).
  • the OPCs are thawed and cultured prior to use.
  • the OPCs are thawed and cultured in suspension prior to use.
  • the OPCs are thawed and cultured on an adherent substrate prior to use.
  • the period for culturing after thawing may be 1 to 24 hours. In some embodiments, the period for culture after thawing may be from 1 to 2 days.
  • the OPCs of the present invention may be held in a suspension culture format.
  • the OPCs of the present invention may be held in a suspension culture format after detachment from a culture plate ( e.g ., post trypsin treatment). For example, adherent OPCs are detached by treatment with trypsin and are transferred to a suspension culture medium.
  • the period of time in which the OPCs are held in suspension may be referred to as the "hold" period.
  • the hold period in suspension is advantageous for at least the following 3 reasons: 1) would allow cells to recover from a potentially damaging enzyme treatment prior to transplantation and/or cryopreservation; 2) it would introduce more flexibility in animal surgery scheduling and 3) would make shipment of ready-to-transplant OPCs to an offsite location (laboratory or clinic) possible.
  • the hold period may be 2, 4, 6, 8, 12, 18, or 24 hours.
  • the hold period may be 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
  • any suitable tissue source may be used to derive the OPCs of this invention.
  • the adult human CNS has been shown to contain oligodendrocyte precursor cells that are capable of proliferating, and which could mature into myelinating oligodendrocytes under the appropriate conditions.
  • the population of cells can be derived from late embryo, juvenile, or adult mammalian CNS tissue, or it may be derived from existing cultures of neural stem cells, as described in Weiss, United States patent 5,750,376 , or Johe, United States patent 5,753,506 .
  • the OPCs may also be obtained from any tissue or cellular source that is capable of giving rise to neural tissue. In one preferred embodiment, the OPCs are human.
  • OPCs may be been isolated from neural or neural-derived cells of several mammalian species including, but not limited to, mice, rats, pigs, non-human primates, and humans.
  • Neural or neural-derived cells may be obtained from embryonic, fetal, post-natal, juvenile, or adult neural tissue, which includes brain and spinal cord.
  • neural or neural-derived cells may be obtained from the cerebral cortex, cerebellum, midbrain, brainstem, spinal cord, and ventricular tissue, as well as areas of the PNS including the carotid body and the adrenal medulla.
  • Other preferred areas include regions in the basal ganglia, preferably the striatum, which consists of the caudate and putamen, or various cell groups such as the globus pallidus, the subthalamic nucleus, the nucleus basalis, the substantia nigra pars compacta, as well as from ventricular tissue found lining CNS ventricles, including the subependyma.
  • the subventricular zone and ventral neuroepithelium are preferred source of OPCs in the adult animal.
  • a population of cells exists within the adult CNS that exhibit stem cell properties in their ability to self-renew and to produce the differentiated mature cell phenotypes of the adult CNS such as oligodendrocytes.
  • These stem cells are found throughout the CNS, particularly in the subventricular region and the dentate gyrus of the hippocampus, and represent a source of neural or neural derived cells from which the target OPCs may be isolated.
  • Neural stem cells have also been isolated from a variety of adult CNS ventricular regions, including the frontal lobe, conus medullaris, thoracic spinal cord, brain stem, and hypothalamus.
  • Growth factor-responsive stem cells can be isolated from many regions of the neuraxis and at different stages of development, of murine, rodent, mammalian, and human CNS tissue. These cells vary in their response to growth factors such as EGF, basic FGF (bFGF, FGF-2) and transforming growth factor alpha (TGF ⁇ ) and can be maintained and expanded in culture in an undifferentiated state for long periods of time.
  • EGF EGF
  • bFGF basic FGF
  • FGF ⁇ transforming growth factor alpha
  • OPCs can be induced to proliferate either by culturing the cells in suspension or on an adherent substrate. See, e.g ., U.S. Pat. Nos. 5,750,376 and 5,753,506 , and medium described therein. Both allografts and autografts are contemplated for transplantation purposes.
  • OPCs of the present embodiments are cultured in a medium that permits their growth and proliferation.
  • the culture in which the isolated OPCs proliferates can be a serum-free medium containing one or more predetermined growth factors effective for inducing proliferation.
  • the culture medium may be supplemented with a growth factor selected from platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2; bFGF), NT3, IGF1 or combinations thereof.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • FGF-2 basic fibroblast growth factor
  • NT3, IGF1 or combinations thereof.
  • the culture medium may be further supplemented with N2 and B27.
  • the conditions in which the OPCs differentiate to oligodendrocytes include culturing the OPC progeny on a laminin or laminin plus fibronectin-coated surface in culture medium containing fetal bovine serum (FBS) or T3 (triiodothyronine) without EGF, bFGF, PDGF, NT3, IGF1 or LIF.
  • FBS fetal bovine serum
  • T3 triodothyronine
  • the OPCs of the present embodiments may be passaged from 1 to 20 times (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 times) post isolation from the primary tissue source and may be induced to proliferate by culturing the cells in suspension or on an adherent substrate.
  • Passaging typically involves detaching cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures and so on. The time between passaging of cells varies and depends on the growth rate.
  • the proliferation medium can be any medium known in the art to induce proliferation of the OPCs without inducing their differentiation.
  • Example 3 provided herein provides an exemplary medium for proliferating the OPCs of the present embodiments.
  • Cell passage or splitting is necessary to maintain cells in exponential growth. Methods for passaging or splitting cells are well known in the art.
  • the OPCs may be passaged using any known method known in the art.
  • progenitor cells differentiate to oligodendrocytes.
  • Differentiation of the cells can be induced by any method known in the art, which include the liberation of inositol triphosphate and intracellular Ca 2+ , liberation of diacyl glycerol, and the activation of protein kinase C and other cellular kinases, and the like.
  • Treatment with phorbol esters, differentiation-inducing growth factors, hormones and other chemical signals can induce differentiation.
  • Differentiation can be induced by growth factor exhaustion, for example, by removal of mitogens, by leaving the cells in culture without media renewal, or by absence of passaging.
  • the induction of proliferation (and differentiation) of the OPCs can be done either by culturing the cells in suspension or on a substrate onto which they can adhere.
  • proliferation and differentiation of OPCs can be induced, under appropriate conditions, in the host in the following combinations: (1) proliferation and differentiation in vitro, then transplantation, (2) proliferation in vitro, transplantation, then further proliferation and differentiation in vivo, (3) proliferation in vitro, transplantation and differentiation in vivo, and (4) proliferation and differentiation in vivo.
  • Proliferation and differentiation in vivo or in situ can involve a non-surgical approach that coaxes OPCs to proliferate in vivo with pharmaceutical manipulation. Such methods involving the transplantation of OPCs are discussed in further detail below.
  • the target OPC populations identified using the methods described herein are useful in a variety of ways, including for drug screening, diagnostics, transplantation, and treatment.
  • the OPCs may be used to reconstitute a host whose cells have been lost through disease or injury. Genetic diseases associated with cells may be treated by genetic modification of autologous or allogeneic OPCs to correct a genetic defect or treat to protect against disease. Alternatively, normal allogeneic OPCs may be transplanted. Diseases other than those associated with cells may also be treated, where the disease is related to the lack of a particular secreted product such as hormone, enzyme, growth factor, or the like.
  • CNS disorders encompass numerous afflictions such as neurodegenerative diseases (e.g . Alzheimer's and Parkinson's), acute brain injury (e.g . stroke, ischemia, head injury, cerebral palsy) and a large number of CNS dysfunctions ( e.g . depression, epilepsy, and schizophrenia).
  • neurodegenerative diseases e.g . Alzheimer's and Parkinson's
  • acute brain injury e.g . stroke, ischemia, head injury, cerebral palsy
  • CNS dysfunctions e.g . depression, epilepsy, and schizophrenia
  • CNS dysfunctions e.g . depression, epilepsy, and schizophrenia.
  • a large number of CNS dysfunctions e.g . depression, epilepsy, and schizophrenia.
  • diseases which include, for example, Alzheimer's Disease, Multiple Sclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease
  • MS Multiple Sclerosis
  • Huntington's Disease Huntington's Disease
  • the target OPC populations may also be used in the isolation and evaluation of factors associated with the differentiation and maturation of cells.
  • the cells may be used in assays to determine the activity of media, such as conditioned media, evaluate fluids for growth factor activity, involvement with dedication of lineages, or the like.
  • the target OPC populations may be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused.
  • the cells will usually be stored in 7.5% DMSO and 4% HSA (human serum albumin). Once thawed, the cells may be expanded by use of growth factors or cells associated with OPC proliferation and differentiation.
  • the target OPC populations obtained from neural cell populations or neural tissue may be introduced ( e.g ., by transplantation) into a mammal, particularly to compensate for lost or dysfunctional oligodendrocytes.
  • the mammal is preferably a human, canine, feline, rodent, sheep, goat, cattle, horse, pig, or non-human primate. Most preferably, the mammal is human.
  • OPCs may be cultured from brain tissues from mammals of any age, including adults, it is preferable to grow neural stem cells using a mammal's own tissue for autologous transplantation. Allogeneic and xenogcneic transplantations are also possible, particularly when the transplantation site is in the brain or eye, where immunologic rejection is less severe due to the blood-brain or blood-retina barrier.
  • the OPCs of the present embodiments are transplanted at a dose of at least on the order of greater than 1x10 20 total nucleated cells, or at least on the order of 10 19 , or 10 18 , or 10 17 , or 10 16 , or 10 15 , or 10 14 , or 10 13 , or 10 12 , or 10 11 , or 10 10 , or 10 9 , or 10 8 , or 10 7 , or 10 6 , or 10 5 cells.
  • the OPCs of the present embodiments may be transplanted at a dose of between 1 X 10 6 to 1 X 10 12 ,1 X 10 6 to 1 X 10 9 , 1 X 10 8 to 1 X 10 10 , 1 X 10 9 to 1 X 10 12 , and 1 X 10 9 to 1 X 10 10 cells.
  • the dosage of cells is prepared in a sealed, pharmaceutical quality vial in a format that is ready to administer to a subject.
  • target OPCs may be transplanted into a mammal and induced to form oligodendrocytes in vivo.
  • target OPC populations may be expanded in culture using established methods, transplanted into the mammal, and contacted in vivo with the oligodendrocyte promoting factor to produce oligodendrocytes.
  • the transplanted OPCs can be expanded again in vivo by administering to the mammal any biological agents known to increase the number of OPCs.
  • the OPCs of the present invention are transplanted in between 1 to 5 days post passaging, preferably between 1 to 2 days past passaging.
  • the OPCs of the present embodiments may be transplanted as clusters or disassociated cell suspensions. Engraftment data (not shown) using cells obtained in this manner are healthy and result in graft containing high number of myelinating oligodendrocytes.
  • the OPCs of the present embodiments may be held in suspension between 10 minutes to 5 days (e . g ., 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days etc.) post passaging in a pharmaceutical quality vial in a format that is ready to administer to a subject.
  • the OPCs of the present embodiments may be held in suspension at a dose of at least on the order of greater than 1x10 20 total nucleated cells, or at least on the order of 10 19 , or 10 18 , or 10 17 , or 10 16 , or 10 15 , or 10 14 , or 10 13 , or 10 12 , or 10 11 , or 10 10 , or 10 9 , or 10 8 , or 10 7 , or 10 6 , or 10 5 cells.
  • the OPCs of the present embodiments may be held in suspension at a dose of between 1 X 10 6 to 1 X 10 12 , 1 X 10 6 to 1 X 10 9 ,1 X 10 8 to 1 X 10 10 , 1 X 10 9 to 1 X 10 12 , and 1 X 10 9 to 1 X 10 10 cells.
  • the dosage of cells is prepared in a sealed, pharmaceutical quality vial in a format that is ready to administer to a subject.
  • the oligodendrocyte promoting factors or the biological agents may be administered by any suitable route established in the art, including, for example, orally, topically, rectally, vaginally, intrathecally, intravascularly, intravenously, intramuscularly, intraperitoneally, transdermally, intradermally, subcutaneously, nasally or by inhalation.
  • the route of administration depends primarily on the nature of the agent.
  • GM-CSF is capable of crossing the blood-brain barrier, hence it can be administered systemically as well as into the brain.
  • the preferred method of administration is injection ( e.g ., with a needle or a catheter) or infusion.
  • the target OPCs may be transplanted "naked" into patients according to conventional techniques, into the CNS as described, for example, in U.S. Pat. Nos. 5,082,670 and 5,618,531 , or into any other suitable site in the body.
  • the OPCs may be transplanted directly into the CNS. Parenchymal and intrathecal sites are contemplated. It will be appreciated that the exact location in the CNS will vary according to the disease state.
  • the OPCs may be allowed to aggregate prior to implantation, or may be applied directly as dissociated single cells.
  • transplantation is preferably performed using small sized aggregates approximately 10-500 ⁇ m in diameter, preferably 40-50 ⁇ m in diameter.
  • Preferably, from about 1 million cells to about 1 billion cells are transplanted. For example, a total of about 1 million, about 5 million, about 10 million, about 25 million, about 50 million, about 75 million, about 100 million, about 250 million, about 500 million, about 750 million, or about 1 billion cells are transplanted.
  • the OPCs are preferably introduced into the brain or spinal cord of the mammal, particularly at sites where oligodendrocytes are insufficient and/or dysfunctional, for example, around axons that have been demyelinated.
  • areas of demyelination are generally associated with plaque like structures, which can be visualized with magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • the cells may also be transplanted into other areas of the central nervous system.
  • One particularly useful approach is to transplant into the "mirror image" location of a target lesion in the other hemisphere, since cells are known to efficiently migrate to the corresponding location in the opposite hemisphere through the corpus callosum.
  • the OPCs may be introduced directly to regions of the brain or spinal cord. Directed introduction of the OPCs may be carried out using any methods known in the art.
  • the OPCs are introducted to the target brain region via injection.
  • the OPCs are introduced into brain regions that are heavily myelinated (rich in white matter).
  • the fimbria is a prominent band of white matter along the medial edge of the hippocampus.
  • White matter forms the bulk of the deep parts of the brain and the superficial parts of the spinal cord.
  • the corpus callosum is the largest white matter structure in the brain that connects the left and right cerebral hemispheres.
  • the target brain regions include the fimbria, callosum, cerebral peduncle, internal capsule, spinal cord, brain stem, motor cortex, olfactory cortex, somatosensory cortex, anterior cingulate gyrus, the Inferior temporal lobe, and the Dorsolateral prefrontal cortex, and medulla oblongata.
  • Aggregates of gray matter such as the basal ganglia (caudate nucleus, putamen, globus pallidus, subthalamic nucleus, nucleus accumbens) and brain stem nuclei (red nucleus, substantia nigra, cranial nerve nuclei) are spread within the cerebral white matter. Such areas are also target brain regions.
  • Target brain regions include, but are not limited to, the telencephalon (cerebral hemispheres, forebrain), diencephalon (thalamus, hypothalamus, epithalamus, prethalamus or subthalamus and pretectum), mesencephalon (midbrain), cerebellum, pons, and medulla oblongata.
  • the mesencephalon includes the tectum (inferior colliculi and superior colliculi) and cerebral peduncle (midbrain tegmentum, crus cerebri, substantia nigra).
  • the substantia nigra is part of the basal ganglia; the other parts of the basal ganglia include the striatum (caudate nucleus, putamen, and nucleus accumbens), globus pallidus, and subthalamic nucleus.
  • Target brain regions may include the brain stem, striatum, internal capsule, caudate nucleus and putamen.
  • the OPCs of the present embodiments may be genetically modified to provide a therapeutically effective biologically active molecule.
  • the genetically modified OPCs may be transplanted or introduced to a subject in need thereof as described above.
  • the OPCs of the present embodiments may be genetically modified to express a particular form of Myelin Proteolipid Protein (PLP), such as in the case of autologous transplant.
  • PGP Myelin Proteolipid Protein
  • the OPCs of the present embodiments may be genetically modified to express one or more of the following: telomerase (to prevent telomere erosion), growth factors, morphogens, enzymes, anti-apoptotic genes (e.g ., sonic hedgehog, FGF2, NT3, BDNF, PDGF, IGF, NGF), arylsuphatase A (metachromatic leukodystrophy), galactosylceramidase (krabbe's), superoxide dismutase and other proteins involved in antioxidant defense, and Bcl-XL.
  • telomerase to prevent telomere erosion
  • growth factors e.g sonic hedgehog, FGF2, NT3, BDNF, PDGF, I
  • the OPCs described herein can be genetically engineered or modified according to known methodology.
  • the term “genetic modification” refers to the stable or transient alteration of the genotype of a cell by intentional introduction of exogenous DNA.
  • DNA may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful DNA sequences.
  • the term “genetic modification” is not meant to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like.
  • a gene of interest i.e., a gene that encodes a biologically active molecule
  • a gene of interest can be inserted into a cloning site of a suitable expression vector by using standard techniques. These techniques are well known to those skilled in the art. See, e.g. , WO 94/16718 , incorporated herein by reference.
  • the expression vector containing the gene of interest may then be used to transfect the desired cell line.
  • Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection, electroporation, biolistics, or viral transfection may be utilized.
  • Commercially available mammalian transfection kits may be purchased from e.g., Stratagene. Human adenoviral transfection may be accomplished as described in Berg et al. Exp. Cell Res., 192, pp. (1991 ). Similarly, lipofectamine-based transfection may be accomplished as described in Cattaneo, Mol. Brain Res., 42, pp. 161-66 (1996 ).
  • host/expression vector combinations may be used to express a gene encoding a biologically active molecule of interest. See, e.g., U.S. Pat. No. 5,545,723 for suitable cell-based production expression vectors.
  • Increased expression of the biologically active molecule can be achieved by increasing or amplifying the transgene copy number using amplification methods well known in the art.
  • amplification methods include, e.g., DHFR amplification (see, e.g., Kaufman et al., U.S. Pat. No. 4,470,461 ) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 , and European published application EP 338,841 ).
  • any expression vector known in the art may be used to express the biologically active molecule.
  • a lentivirally-derived vector may be particularly useful for the delivery of exogenous genes.
  • exogenous genes may need to be introduced into the target OPCs expression.
  • Such genes may be under the control of a constitutive or inducible promoters to effect optimal co-expression.
  • Exogenous DNA may be introduced to a precursor cell by viral vectors (retrovirus, modified herpes viral, herpes-viral, adenovirus, adeno-associated virus, lentivirus and the like) or direct DNA transfection (lipofection, CaPO 4 transfection, DEAE-dextran, electroporation, and the like).
  • viral vectors retrovirus, modified herpes viral, herpes-viral, adenovirus, adeno-associated virus, lentivirus and the like
  • direct DNA transfection lipofection, CaPO 4 transfection, DEAE-dextran, electroporation, and the like.
  • OPCs of the present embodiments may be genetically modified for drug screening purposes or for the purposes of detecting cells of the oligodendrocyte or OPC lineage.
  • OPCs may be genetically modified with one or more reporter genes.
  • reporter genes include fluorescent proteins (e.g ., green fluorescent proteins, yellow fluorescent proteins, blue fluorescent proteins, cyan fluorescent proteins, etc.), DsRed2, mCherry, tdTomato, and AmCyan1.
  • Preferred promoters include one or more of the following promoters: MBP, CNPasc, Olig2, Sox10, Plp, and PDGFR promoter.
  • a number of neurologic diseases are associated with defects in myelination and in neuronal homeostasis and function.
  • demyelinating diseases or conditions or dysmyelinating disorders include, but are not limited to, multiple sclerosis (including the relapsing and chronic progressive forms of multiple sclerosis, acute multiple sclerosis, neuromyelitis optica (Devic's disease)), diffuse cerebral sclerosis (including Shilder's encephalitis periaxialis diffusa and Balo's concentric sclerosis).
  • Demyelinating diseases also include a variety of diseases wherein demyelination is caused by viral infections, vaccines, spinal cord injury, and genetic disorders.
  • demyelinating diseases or dysmyelinating disorders include, but are not limited to, acute disseminated encephalomyelitis (occurring after measles, chickenpox, rubella, influenza or mumps; or after rabies or smallpox vaccination), necrotizing hemorrhagic encephalitis (including hemorrhagic leukoencephalitis), and leukodystrophies (including Krabbe's globboid leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, adrenomyeloneuropathy, adrenomyeloneuropathy, radiation induced myelination disorders, transverse myelitits, Pelizaeus-Merzbacher disease, Canavan's disease and Alexander's disease).
  • acute disseminated encephalomyelitis occurring after measles, chickenpox, rubella, influenza or mumps; or after rab
  • the demyelinating disease or dysmyelinating disorders is preferably multiple sclerosis, cerebral palsy, diffuse cerebral sclerosis, or Pelizaeus-Merzbacher disease (PMD), and, most preferably, Pelizaeus-Merzbacher disease.
  • PMD Pelizaeus-Merzbacher disease
  • the cells and methods of this invention may be useful in the treatment of various neurodegenerative diseases, demyelinating diseases and/or dysmyelinating disorders. It is contemplated that the cells will replace diseased, damaged or lost tissue in the host. Alternatively, the transplanted tissue may augment the function of the endogenous affected host tissue.
  • oligodendrocytes are capable of myelinating (or remyelinating) demyelinated neurons in the mammal, whereby dysmyelinating disorders and/or demyelinating diseases in the mammal can be treated or ameliorated.
  • oligodendrocytes are capable of myelinating (or remyelinating) demyelinated neurons in the mammal, whereby dysmyelinating disorders and/or demyelinating diseases in the mammal can be treated or ameliorated.
  • oligodendrocyte production in vivo by identifying and isolating target populations of OPCs, culturing the target populations of OPCs under conditions to promote their proliferations, differentiating the target populations of OPCs into oligodendrocytes, and administering the oligodendrocytes to a mammal under conditions that result in oligodendrocyte engraftment.
  • the resultant oligodendrocytes are capable of myelinating (or remyelinating) demyelinated neurons in the mammal, whereby dysmyelinating disorders and/or demyelinating diseases in the mammal can be treated or ameliorated.
  • oligodendrocyte production in vivo by identifying and isolating target populations of OPCs and administering target OPCs to a mammal under conditions that result in oligodendrocyte engraftment.
  • the resultant oligodendrocytes are capable of myclinating (or remyelinating) demyelinated neurons in the mammal, whereby dysmyelinating disorders and/or demyelinating diseases in the mammal can be treated or ameliorated.
  • the OPCs of the present invention may also be used in a method of drug screening or drug discovery. Any cell-based drug screening protocol known in the art may be used in conjunction with the OPCs of the present invention.
  • a wide variety of assays may be used for this purpose, including toxicology testing; inumunoassays for protein binding; determination of cell growth, differentiation and functional activity; production of hormones; and the like.
  • the assays may be performed in vitro , in situ , in vivo , and ex vivo .
  • the OPCs of the present invention may be used in a drug screening method comprising the steps of a) selecting from an enriched target OPC population, b) engrafting a non-human mammal with the resulting enriched population; c) administering a test compound to the non-human mammal; and d) comparing the effect of administration of said test compound in the engrafted mammal with a control non-human mammal not administered said test compound.
  • the present invention provides an in vitro method of screening for compounds that affect a biological function of an enriched population of target oligodendrocyte precursor cells comprising: (a) contacting an enriched population of target oligodendrocyte precursor cells obtained by the method of claim 1 with a test compound; and (b) detecting a change in a biological function of the oligodendrocyte precursor cells.
  • the change in biological function may include, but is not limited to, changes in one or more of the following: myelination, differentiation into oligodendrocytes, proliferation rate, cell migration, cell viability, gene expression, protein expression, protein levels in the culturing medium, dedifferentiation, growth characteristics, and/or cell morphology.
  • Agents are screened for biological activity by adding the agent to at least one and usually a plurality of cell samples.
  • the change in parameters in response to the agent is measured, and the result evaluated by comparison to reference cultures, e.g . in the presence and absence of the agent, obtained with other agents, etc.
  • the agents may be conveniently added in solution, or readily soluble form, to the medium of cells in culture.
  • the agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, by adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added.
  • the first fluid is passed over the cells, followed by the second.
  • a bolus of the test compound is added to the volume of medium surrounding the cells.
  • the overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.
  • a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the parameter with high affinity fluorescent moieties are readily available for labeling virtually any biomolccule, structure, or cell type.
  • Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered to autofluoresce, e.g, by expressing them as green fluorescent protein chimeras inside cells.
  • antibodies can be modified to provide a fluorescent dye as part of their structure.
  • parameters may be measured using other than fluorescent labels, using such immunoassay techniques as radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques.
  • RIA radioimmunoassay
  • ELISA enzyme linked immunosorbance assay
  • the quantization of nucleic acids, especially messenger RNAs is also of interest as a parameter. These can be measured by hybridization techniques that depend on the sequence of nucleic acid nucleotides. Techniques include polymerase chain reaction methods as well as gene array techniques.
  • any encapsulation protocol known in the art may be used with the OPCs of the present invention.
  • the OPCs of the present invention may be encapsulated and used to deliver biologically active molecules, according to known encapsulation technologies, including microencapsulation (see, e.g. , U.S. Patents 4,352,883 ; 4,353,888 ; and 5,084,350 macroencapsulation ( see, e.g. , U.S. Patents 5,284,761 , 5,158,881 , 4,976,859 and 4,968,733 and published PCT patent applications WO 92/19195 , WO 95/05452 .
  • OPCs are encapsulated, macroencapsulation as described in U.S. Patents 5,284,761 ; 5,158,881 ; 4,976,859 ; 4,968,733 ; 5,800,828 and published PCT patent application WO 95/05452 , is preferred.
  • Cell number in the devices can be varied. Preferably each device contains between 10 3 -10 9 cells, most preferably 10 5 -10 7 cells.
  • a large number of macroencapsulation devices may be implanted in the patient; preferably between one to 10 devices.
  • Cells were ressuspended in Hank's balanced salt solution containing 1mM Sodium Pyruvate and 0.1% human serum albumin (staining buffer) and stained with the CD133 antibody.
  • CD133 + cells were aseptically sorted using a BD Vantage flow cytometer, under enrichment mode.
  • CD133 + enriched fraction was centrifuged, ressuspended in staining buffer and incubated with a 1:100 dilution of rabbit anti PDGFR ⁇ polyclonal antibody (IgG) for 2 hours at 4° C.
  • IgG rabbit anti PDGFR ⁇ polyclonal antibody
  • PDGFR ⁇ labeled cells were incubated for polyclonal goat anti-rabbit IgG-FITC antibody (Caltag).
  • PDGFR ⁇ positive (FITC labeled) cells were aseptically sorted using a BD Vantage flow cytometer, under purity mode. Sorted cells were cultured on poly-L-ornithine, laminin and fibronectin coated culture flasks in DMEM medium supplemented with B27, N2, NAC, L-Glutamine, Na Pyruvate, FGF2, PDGF-AA and NT3 (complete medium), with or without IGF 1. Cell passaging was achieved by mild trypsin treatment and re-plating in the same medium.
  • An alternative method used for the purification of PDGFR ⁇ positive cells is to stain total brain cells (with or without enrichment for CD 133) with the mouse monoclonal antibody 1:50 PDGFR ⁇ -PE (Pharmingen) for 2h at 4° C, followed by purity aseptic sorting using a BD Aria flow cytometer.
  • CD105 - cells as a selection marker is based on the present inventors discovery that some cultures of purified PDGFR ⁇ + cells expand at a much faster rate then others. Such accelerated growth was generally accompanied by the appearance of a cell type with a morphology distinct from that of oligodendrocyte progenitors. Based on the morphology and accelerated growth in culture, it is likely that these cells are PDGFR ⁇ + fibroblasts; the growth advantage of fibroblasts in FGF2 containing media is well documented.
  • fibroblasts may also negatively condition the culture medium, affecting the growth kinetics and the differentiation process of oligodendrocyte progenitors into oligodendrocytes, both in vitro and in vivo. The presence of these fibroblastic cells therefore reduces the efficiency in obtaining an expanded culture of oligodendrocyte progenitors.
  • oligodendrocyte progenitor population It is desirable to define a more pure and homogeneous oligodendrocyte progenitor population, especially in the case when contaminating cells have growth advantage. Therefore a search was initiated for cell surface markers expressed specifically in fibroblasts that could be used to distinguish them from PDGFR ⁇ + oligodendrocyte progenitors. A panel of antibodies was used to stain cultures containing a mixture of fibroblasts and oligodendrocytes.
  • the sorting protocol for the isolation of fetal derived human oligodendrocyte progenitors includes two antibodies, CD105-APC and PDGFR ⁇ -PE. Various cell lots have been generated using this two antibody protocol. The results indicate that these cell lots have similar growth characteristics and oligodendrocyte differentiation potential. Further, the appearance of fibroblasts is not observed in these cultures, up to passage 15, the highest passage tested. The results demonstrate that CD 105 is a useful negative selection marker for use in obtaining a desirable population of oligodendrocyte progenitors.
  • Proliferation medium was prepared with the following components in the indicated concentrations: Component Final Concentration DMEM, glutamine (Invitrogen, cat# 25030-081) 2 mM, Na Pyruvate (Sigma, cat# S8636), 1mM, NAC (Sigma, cat# A9165), 1mM, N2 supplement (Invitrogen, cat# 17502-048; containing transferrin, insulin, putrescine, selenium and progesterone), B27 supplement (Invitrogen, cat # 17504-044), 20 ng/ml human bFGF (Biosource, cat# PHG0024), 20 ng/ml PDGF-AA (Peprotech, cat# 100-13A) 10ng/ml NT3 (Peprotech, cat# 450-03), 100ng/ml IGF1 (Peprotech, cat #AF-100-11).
  • Component Final Concentration DMEM glutamine (Invitrogen, cat# 25030-081) 2 mM, Na Pyr
  • proliferating OPCs are induced to differentiate by physical removal or exhaustion of the growth factor mitogens from the cell culture with addition of triiodothyronine (T3).
  • T3 triiodothyronine
  • the staining protocol for oligodendrocytes was as follows:
  • Cells are incubated with secondary antibodies (donkey anti mouse IgG/Alexa488 used at 1:500, (Invitrogen, Cat# A21202); or goat anti mouse IgM/Alexa 488, (Invitrogen, Cat# A21042) diluted in 1% HS for 1hr at room temperature in the dark. Preparations are washed twice for 5 min with 0.1 M PBS in the dark. Preparations are mounted onto slides face down with mounting medium (Vectashield Mounting Medium, Vector Laboratories, cat# H-1000) or left on culture wells for quantification and qualification of staining and stored at 4° C.
  • secondary antibodies donkey anti mouse IgG/Alexa488 used at 1:500, (Invitrogen, Cat# A21202); or goat anti mouse IgM/Alexa 488, (Invitrogen, Cat# A21042) diluted in 1% HS for 1hr at room temperature in the dark. Preparations are washed twice for 5 min with 0.1 M PBS
  • stain with Hoechst may be used as follows. Cells prepared as above are washed with Hoechst solution (diluted 1:10,000 in 0.1% saponin, Sigma, Cat#S4521). Cells are incubated in Hoechst solution for 5 min at room temperature, followed by 2 washes in 0.1M PBS.
  • the OPCs are induced to differentiate by removal of the growth factor mitogens and provision of 1% serum.
  • This differentiation protocol produces cell cultures highly enriched in oligodendrocytes.
  • the OPCs are induced to differentiate by removal of the growth factor mitogens and provision of 30nM T3 (Sigma, cat# T5516).
  • This differentiation protocol produces cell cultures highly enriched in oligodendrocytes.
  • the hollow fibers are fabricated from a polyether sulfone (PES) with an outside diameter of 720 m and a wall thickness of a 100 m (AKZO-Nobel Wuppertal, Germany). These fibers are described in U.S. Pat. Nos. 4,976,859 and 4,968,733 , herein incorporated by reference.
  • the fiber may be chosen for its molecular weight cutoff.
  • a PES#5 membrane which has a MWCO of about 280 kd is occasionally used. In other studies, a PES#8 membrane which has a MWCO of about 90 kd may be used.
  • the devices typically comprise: 1) a semipermeable poly (ether sulfone) hollow fiber membrane fabricated by AKZO Nobel Faser AG; 2) a hub membrane segment; 3) a light cured methacrylate (LCM) resin leading end; and 4) a silicone tether.
  • the semipermeable membrane used typically has the following characteristics: Internal Diameter 500 + 30 m Wall Thickness 100 + 15 m Force at Break 100 + 15 cN Elongation at Break 44 + 10% Hydraulic Permeability 63 + 8 (ml/min m 2 mmHg) nMWCO (dextrans) 280 + 20 kd.
  • the components of the device are commercially available.
  • the LCM glue is available from Ablestik Laboratories (Newark, Del.); Luxtrak Adhesives LCM23 and LCM24).
  • the tether material is available from Specialty Silicone Fabricators (Robles, Calif.).
  • the tether dimensions are 0.79 mm ODX0.43 mm IDXlength 202 mm.
  • the morphology of the device is as follows:
  • Fiber material is first cut into 5 cm long segments and the distal extremity of each segment sealed with a photopolymerized acrylic glue (LCM-25, ICI). Following sterilization with ethylene oxide and outgassing, the fiber segments are loaded with a suspension of between 10 4 -10 7 cells, either in a liquid medium, or a hydrogel matrix (e.g ., a collagen solution (Zyderm TM ), alginate, agarose or chitosan) via a Hamilton syringe and a 25 gauge needle through an attached injection port. The proximal end of the capsule is sealed with the same acrylic glue.
  • a photopolymerized acrylic glue (LCM-25, ICI). Following sterilization with ethylene oxide and outgassing, the fiber segments are loaded with a suspension of between 10 4 -10 7 cells, either in a liquid medium, or a hydrogel matrix (e.g ., a collagen solution (Zyderm TM ), alginate, agarose or chitosan) via a
  • a silicone tether (Specialty Silicone Fabrication, Taunton, Ma.) (ID: 690 m; OD: 1.25 mm) is placed over the proximal end of the fiber allowing easy manipulation and retrieval of the device.
  • Target OPCs may be transplanted into rodent brain to assess graft viability, integration, phenotypic fate of the grafted cells, as well as behavioral changes associated with grafted cells in healthy animals.
  • Transplantation is performed according to standard techniques. For example, adult rats are anesthetized with sodium pentobarbitol (45 mg/kg, i.p.) and positioned in a Kopf stereotaxic instrument. A midline incision is made in the scalp and a hole drilled for the injection of cells. Rats receive implants of target OPCs into the striatum using a glass capillary attached to a 10 ⁇ l Hamilton syringe. Each animal receives a total of about 250,000-500,000 cells in a total volume of 2 ⁇ l. Cells are transplanted 1-2 days after passaging and the cell suspension is made up of undifferentiated OPC clusters of 5-20 cells. Following implantation, the skin is sutured closed.
  • sodium pentobarbitol 45 mg/kg, i.p.
  • Target OPCs may be transplanted into rodent brain to assess graft viability, integration, phenotypic fate of the grafted cells, as well as behavioral changes associated with grafted cells in lesioned or diseased animals.
  • Transplantation is performed according to standard techniques. For example, newborn md rats or jimpy mice are anesthetized by hypothermia and positioned in a Kopf stereotaxic instrument. A midline incision is made in the scalp and a hole drilled for the injection of cells. Animals receive implants of target OPCs into the corpus callosum, fimbria, cerebellar peduncle and/or spinal cord using a glass capillary attached to a 10 ⁇ l Hamilton syringe. Each animal receives a total of about 300,000-600,000 cells in a total volume of 6 ⁇ l. Cells are transplanted immediately or 1-2 days after passaging and the cell suspension is made up of undifferentiated single OPCs or clusters of 5-20 cells. Following implantation, the skin is sutured or staple closed.
  • mice are anesthetized by hypothermia or isofluorane and positioned in a Kopf stereotaxic instrument. A midline incision is made in the scalp and a hole drilled for the injection of cells.
  • Mice receive implants of target OPCs into the corpus callosum, fimbria, cerebellar peduncle and/or spinal cord using a glass capillary attached to a 10 ⁇ l Hamilton syringe. Each animal receives a total of about 300,000-600,000 cells in a total volume of 6 ⁇ l.
  • Cells are transplanted immediately or 1-2 days after passaging and the cell suspension is made up of undifferentiated single OPCs or clusters of 5-20 cells. Following implantation, the skin is sutured or staple closed.
  • Example 9 Treatment of Neurodegenerative Disease Using Progeny of Target OPCs In Vitro
  • Target OPCs are obtained from fetal brain tissue following routine suction abortion which is collected into a sterile collection apparatus. A 2x4x1 mm piece of tissue is dissected and dissociated as in Examples 1 or 2. Target OPCs are then proliferated. The target OPC progeny are used for neurotransplantation into a blood-group matched host with a neurodegenerative disease. Surgery is performed using a BRW computed tomographic (CT) stereotaxic guide. The patient is given local anesthesia suppiemencea with intravenously administered midazolam. The patient undergoes CT scanning to establish the coordinates of the region to receive the transplant.
  • CT computed tomographic
  • the injection cannula consists of a 17-gauge stainless steel outer cannula with a 19-gauge inner stylet. This is inserted into the brain to the correct coordinates, then removed and replaced with a 19-gauge infusion cannula that has been preloaded with 30 ⁇ l of tissue suspension. The cells are slowly infused at a rate of about 3 ⁇ l/min as the cannula is withdrawn. Multiple stereotactic needle passes are made throughout the area of interest, approximately 4 mm apart. The patient is examined by CT scan postoperatively for hemorrhage or edema. Neurological evaluations are performed at various post-operative intervals, as well as PET scans to determine metabolic activity of the implanted cells.
  • Target OPC progeny are propagated as described herein.
  • the cells are then transfected using a calcium phosphate transfection technique.
  • the cells are mechanically dissociated into a single cell suspension and plated on tissue culture-treated dishes at 50% confluence (50,000-75,000 cells/cm 2 ) and allowed to attach overnight.
  • the modified calcium phosphate transfection procedure is performed as follows: DNA (15-25 ⁇ g) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA, pH 7.5) diluted to 440 ⁇ l with TE, and 60 ⁇ l of 2M CaCl 2 (pH to 5.8 with 1M HEPES buffer) is added to the DNA/TE buffer. A total of 500 ⁇ l of 2XHeBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na 2 HPO 4 , 12 mM dextrose, 40 mM HEPES buffer powder, pH 6.92) is added dropwise to this mix.
  • 2XHeBS HPES-Buffered saline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na 2 HPO 4 , 12 mM dextrose, 40 mM HEPES buffer powder, pH 6.92
  • the mixture is allowed to stand at room temperature for 20 minutes.
  • the cells are washed briefly with 1X HeBS and 1 ml of the calcium phosphate precipitated DNA solution is added to each plate, and the cells are incubated at 37 ° for 20 minutes. Following this incubation, 10 mls of complete medium is added to the cells, and the plates are placed in an incubator (37 ° C., 9.5% CO 2 ) for an additional 3-6 hours.
  • the DNA and the medium are removed by aspiration at the end of the incubation period, and the cells are washed 3 times with complete growth medium and then returned to the incubator.
  • Target OPCs are proliferated as described herein and then infected with lentiviral vectors containing genes of interest in addition to a report gene such as GFP (green fluorescence protein).
  • Lentivirus suspensions are added to the culture medium where OPCs are proliferated and incubated for 24 hours. After 24h, the culture medium is removed and replaced with fresh medium and the OPCs are cultured for another 3 days. Cells are collected and centrifuged and cells expressing the gene of interest are sorted by flow cytometry. Positive cells are returned to the proliferative medium.
  • the transduced OPC progeny are transplanted into a rodent or human patient using the procedures described in the previous Examples.
  • Example 12 Transplantation of OPCs into rodent models of spinal cord injury
  • Target OPCs may be transplanted into rodent spinal cord to assess graft viability, integration, phenotypic fate of the grafted cells, as well as behavioral changes associated with grafted cells in spinal cord lesioned animals.
  • Animals receive a laminectomy at vertebral level T9. Animals then receive a 50-kilodyne (kd) contusion spinal cord injury using an Infinite Horizon Impactor (Precision Systems and Instrumentation, Lexington, KY). Seven days after spinal cord injury, mice are tested using the Basso, Beattie, and Bresnahan (BBB) rating scale and randomized to receive OPCs or vehicle control. Cells are injected bilaterally anterior and posterior to the epicenter of the lesion using a beveled grass micropipette affixed to a nanoinjector device. Each animal receives 50,000 to 80,000 cells.
  • kd 50-kilodyne
  • Infinite Horizon Impactor Precision Systems and Instrumentation, Lexington, KY
  • BBB Bresnahan
  • Myelin oligodendrocyte glycoprotein (MOG)-induced murine experimental autoimmune encephalomyelitis (EAE) is a widely accepted model for studying the clinical and pathological features of multiple sclerosis.
  • Transplantation is performed according to standard techniques. For example, adult animals affected by MOG-induced EAE are anesthetized using isoflurane gas and positioned in a Kopf stereotaxic instrument. A midline incision is made in the scalp and a hole drilled for the injection of cells. Animals receive implants of target OPCs into the corpus callosum, fimbria, cerebellar peduncle, lateral ventricular space and/or spinal cord using a glass capillary attached to a 10 ⁇ l Hamilton syringe. Each animal receives a total of about 300,000-600,000 cells in a total volume of 6 ⁇ l. Cells are transplanted immediately or 1-2 days after passaging and the cell suspension is made up of undifferentiated single OPCs or clusters of 5-20 cells. Following implantation, the skin is sutured or staple closed.
  • shiverer mouse In order to determine whether FACS-isolated, in vitro expanded oligodendrocyte progenitor cells survive, migrate and are capable of in vivo myelination, a series of transplantation studies were conducted using the shiverer mouse, a rodent model of dysmyelination.
  • the shiverer mouse contains a naturally occurring deletion of a large portion of the mbp gene which results in incomplete CNS myelin formation.
  • the shiverer mice were backcrossed to the immunodeficient NOD-Scid mouse.
  • Engraftment of oligodendrocyte progenitors was studied in two different Shiverer/Scid age groups: as juveniles (P21-P30) and as neonates (P0-P1). Shiverer/Scid mice have a relatively short lifespan of around 8 weeks and therefore the longest post transplantation time point studied was about 8 weeks in the case of neonatal injections and 5 weeks for juvenile injections.
  • Oligodendrocyte progenitors are grown as a monolayer.
  • OPCs were lifted off the flasks using trypsin, following a protocol similar to that used to passage OPCs.
  • Cells were then exposed to trypsin inhibitor to stop the proteolytic digestion, washed twice in culture medium and ressuspended in ex - vivo medium containing the antioxidant NAC (1mM) at a final density of 1E5 cells/ ⁇ l.
  • Juvenile or neonatal shiverer/Scid mice were placed in a stereotaxic frame and 1 ⁇ l of OPC suspension was injected into 2-3 brain locations, bilaterally (4-6 total injections/mouse). Injections targeted the corpus callosum, fimbria and the cerebellar peduncle (see figure 1 ), regions of the brain that are heavily myelinated (rich in white matter) in myelin-competent animals but severely hypomyelinated in the shiverer/scid mouse.
  • mice were sacrificed at different time points, up to 8 weeks, and their brains analyzed for the presence of human cells using the monoclonal antibody SC121 and for the presence of human derived oligodendrocytes, capable of myelinating mouse axons, using an MBP antibody. Because shiverer mice have a mutation that deletes most of the mbp gene, MBP protein is not produced by mouse oligodendrocytes and therefore any MBP detected by the anti-MBP antibody is of human origin.
  • Donor ID age Passage tested Shiverer age group Total # transplanted Shi / Scid mice Presence of donor cells SC121 + Myelin production MBP + 2657, 18 wks 3, 13 & 14 Juvenile 10 10/10 10/10 2703, 18 wks 6, 9, 14, 15 & 16 Juvenile & Neonates 4 juv + 15 neo 19/19 19/19 2710, 18 wks 2 Neonates 2 2/2 2/2 2711, 18 wks 4,6, 11 & 12 Neonates 16 16/16 16/16 16/16

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Claims (21)

  1. Angereicherte Population von Oligodendrozyten-Vorläuferzellen (OPC), die von neuralen oder neural abgeleiteten Zellen abgeleitet sind, wobei die Population mit OPC angereichert ist, die PDGFRα+ und CD105- und A2B5lo/- sind, wobei mindestens 30 % der Zellen in der Population PDGFRα+, CD105- und A2B5lo/- sind, wobei die OPC in der Population zur Differenzierung in mature Oligodendrozyten unter Bedingungen gebracht werden, die eine Differenzierung zulassen.
  2. OPC-Population nach Anspruch 1, wobei mindestens 50 % oder mindestens 70 % oder mindestens 90 % der Zellen in der Population PDGFRα+, CD105- und A2B5lo/- sind.
  3. In-vitro-Verfahren zur Herstellung einer mit Oligodendrozyten-Vorläuferzellen (OPC) aus neuralen oder neural abgeleiteten Zellen angereicherten Zellpopulation, umfassend
    (a) Inkontaktbringen der neuralen oder neural abgeleiteten Zellen mit mindestens einem Antikörper, der an CD105 bindet, und
    (b) Entfernen von Zellen, die an den Antikörper binden, der an CD105 bindet, und
    (c) Inkontaktbringen der übrigen Zellen mit mindestens einem Antikörper, der an PDGFRα bindet, wobei die OPC PDGFRα+ und CD105- sind, wobei die Zellpopulation angereichert wird, um mindestens 30 % OPC zu enthalten, die PDGFRα+, CD105- und A2B5lo/- sind, wobei die OPC in der Population zur Differenzierung in mature Oligodendrozyten unter Bedingungen gebracht werden, die eine Differenzierung zulassen.
  4. Verfahren nach Anspruch 3, weiter umfassend den Schritt des Inkontaktbringens der übrigen neuralen oder neural abgeleiteten Zellen im Anschluss an Schritt (b) mit mindestens einem Antikörper, der an einen Zelloberflächenmarker bindet, der aus der Gruppe ausgewählt ist, bestehend aus CD45, A2B5, PSA-NCAM, 04 und CD24 und Entfernen der Zellpopulation, die vor Schritt (c) an den Antikörper bindet.
  5. Verfahren zur Steigerung der Anzahl an Oligodendrozyten-Vorläuferzellen (OPC) in einer Zellkultur, umfassend das Kultivieren einer Zellpopulation, umfassend mindestens 30 % OPC zur Steigerung der OPC-Zahl in der Zellkultur, wobei die OPC PDGFRα+, A2B5lo/- und CD105- sind, wobei die OPC in der Population zur Differenzierung in mature Oligodendrozyten unter Bedingungen gebracht werden, die eine Differenzierung zulassen.
  6. Population nach Anspruch 1 oder das Verfahren nach den Ansprüchen 3 oder 5, wobei die OPC auch PSA-NCAMlo/- sind, oder auch CD133+ sind.
  7. OPC-Population nach Anspruch 1 oder das Verfahren nach den Ansprüchen 3 oder 5, wobei die OPC PDGFRα+, CD105-, CD133+ und A2B5-; oder PDGFRα+, CD105-, CD133+, A2B5lo/- und PSA-NCAM- sind.
  8. Verfahren nach Anspruch 5, weiter umfassend das Inkontaktbringen der OPC mit einer wirksamen Menge von mindestens einem biologischen Mittel, das zur Steigerung der OPC-Zahl fähig ist, wobei das biologische Mittel den Leukämie-Inhibitionsfaktor (LIF), den epidermalen Wachstumsfaktor (EGF), den basischen Fibroblasten-Wachstumsfaktor (FGF-2; bFGF), IGF-1, NT3, Shh, CTNF, PDGF, PDGF-AA, N2-Zusatz, B27-Zusatz, DMEM, Glutamin, Natriumpyruvat, N-Acetyl-L-Cystein, Transferrin, Insulin, Putrescin, Selen, Progesteron oder Kombinationen davon umfasst.
  9. Verfahren zur Proliferation von Oligodendrozyten-Vorläuferzellen (OPC), wobei in Gegenwart von eine Differenzierung induzierenden Bedingungen die Zellen Nachkommenzellen produzieren, die in Oligodendrozyten differenzieren, wobei das Verfahren Folgendes umfasst:
    Proliferation einer OPC-Population in serumfreiem Kulturmedium, das einen oder mehr prädeterminierte(n) Wachstumsfaktor(en) enthält, der/die zum Induzieren der OPC-Proliferation wirksam ist/sind, wobei der prädeterminierte Wachstumsfaktor den Leukämie-Inhibitionsfaktor (LIF), epidermalen Wachstumsfaktor (EGF), basischen Fibroblasten-Wachstumsfaktor (FGF-2, bFGF), IGF-1, NT3, Shh, CTNF, PDGF, PDGF-AA oder Kombinationen davon umfasst und wobei die Population OPC umfasst, die PDGFRα+, CD105- und A2B5lo/- sind.
  10. Verfahren nach Anspruch 9, wobei die OPC auch PSA-NCAMlo/- sind oder auch CD133+ sind.
  11. Verfahren nach Anspruch 9, weiter umfassend das Unterziehen der OPC Kulturbedingungen, die eine Oligodendrozyten-Differenzierung zum Hervorrufen einer Differenzierung und differenzierter Oligodendrozyten induzieren.
  12. Angereicherte OPC-Population nach Anspruch 1 zur Verwendung bei der Behandlung einer demyelinisierenden Erkrankung oder dysmyelinisierenden Störung bei einem Säuger.
  13. Angereicherte OPC-Population zur Verwendung nach Anspruch 12, wobei der Säuger ein Mensch ist.
  14. Angereicherte OPC-Population zur Verwendung nach Anspruch 12, wobei die OPC zur Verabreichung an den Säuger mittels Zelltransplantation vorgesehen sind.
  15. Angereicherte OPC-Population zur Verwendung nach Anspruch 12, wobei die Krankheit oder Erkrankung eine Multiple Sklerose, eine diffuse Zerebralsklerose, die Pelizaeus-Merzbacher-Krankheit, eine Zerebralparese, strahleninduzierte Myelinisierungsstörungen, eine akute disseminierte Enzephalomyelitis, eine nekrotisierendhämorrhagische Enzephalitis, Leukodystrophien, eine transverse Myelitis, eine genetisch bedingte demyelinisierende Erkrankung, eine Rückenmarkverletzung, eine Virus-induzierte Demyelinisierung, eine progressive multifokale Leukoenzephalopathie, eine humane T-Zelllymphotrophe Virus I(HTLVI)-assoziierte Myelopathie oder eine ernährungsbedingte Stoffwechselstörung ist.
  16. In-vitro-Verfahren zum Screening auf Verbindungen, die sich auf eine biologische Funktion einer angereicherten Population von Oligodendrozyten-Vorläuferzellen auswirken, umfassend: (a) Inkontaktbringen der angereicherten Population von Oligodendrozyten-Vorläuferzellen nach Anspruch 1 mit einer Testverbindung; und (b) Nachweis einer Veränderung einer biologischen Funktion der Oligodendrozyten-Vorläuferzellen.
  17. Verfahren nach Anspruch 16, wobei die Oligodendrozyten-Vorläuferzellen PDGFRα+, CD105-, A2B5lo/-, PSA-NCAMlo/- und CD133+; oder PDGFRα+, CD105-, A2B5- und CD133+; oder PDGFRα+, CD105-, A2B5lo/-, PSA-NCAM- und CD133+ sind.
  18. Verfahren nach Anspruch 16, wobei die Veränderung in mindestens einem der Charakteristika auftritt, die aus der Gruppe ausgewählt sind, bestehend aus Myelinisierung, Differenzierung in Oligodendrozyten, Proliferationsrate, Zellmigration, Lebensfähigkeit, Genexpression, Proteinexpression, Proteinspiegeln im Kulturmedium, Dedifferenzierung, Wachstumscharakteristika und Zellmorphologie.
  19. In-vitro-Verfahren zum Herstellen einer Zellpopulation, die mit Oligodendrozyten-Vorläuferzellen angereichert ist, umfassend: (a) Inkontaktbringen einer Population von neuralen oder neural abgeleiteten Zellen mit einem Antikörper, der spezifisch an PDGFRα bindet; (b) Auswählen der Zellen aus der Population, die an den Antikörper binden und (c) Eliminieren der Zellen, die CD105+ sind, wobei die ausgewählten Zellen PDGFRαhi sind, wobei die ausgewählten Zellen im Vergleich zu der Population von neuralen oder neural abgeleiteten Zellen mit Oligodendrozyten-Vorläuferzellen angereichert sind, und wobei die Oligodendrozyten-Vorläuferzellen A2B5lo/- sind und unter Bedingungen in mature Oligodendrozyten differenzieren, die eine Differenzierung zulassen.
  20. Verfahren nach Anspruch 19, wobei die neuralen oder neural abgeleiteten Zellen aus einer Neurosphärenkultur oder Adhärenzkultur erhalten werden.
  21. Verfahren nach Anspruch 19, wobei das Verfahren weiter den Schritt zum Eliminieren der Zellen umfasst, die PDGFRαlo/med sind.
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US20180187148A1 (en) 2018-07-05
US10400214B2 (en) 2019-09-03
US20100158878A1 (en) 2010-06-24
EP3187581A1 (de) 2017-07-05
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